CN106929477B - Anti-prostaglandin F2αSpecific monoclonal antibody hybridoma cell strain WXX-2 and application thereof - Google Patents

Abstract

A specific monoclonal antibody against prostaglandin F _2α hybridoma cell line WXX-2 and applications thereof belong to the technical field of clinical detection. The anti-prostaglandin F _2α -specific monoclonal antibody hybridoma cell line WXX-2 of the present invention has been deposited in the General Microbiology Center of the China Microorganism Culture Collection and Administration Commission, and the deposit number is CGMCC No.13087. The anti-prostaglandin F _2α -specific monoclonal antibody is secreted and produced by the anti-prostaglandin F _2α -specific monoclonal antibody hybridoma cell line WXX-2. The hybridoma cell line WXX-2 can secrete and produce a monoclonal antibody with better specificity and higher sensitivity to prostaglandin F _2α , and the 50% inhibitory concentration IC ₅₀ to prostaglandin F _2α is 1.80 μg/L, Can be used for specific detection of prostaglandin F _2α in clinical assays.

Description

A specific monoclonal antibody against prostaglandin F2α hybridoma cell line WXX-2 and its application

technical field

The present invention is a hybridoma cell line WXX-2 with a specific monoclonal antibody against prostaglandin F _2α and its application, and relates to the hybridoma cell line of prostaglandin F _2α and the anti-specific monoclonal antibody produced by the hybridoma cell line, belonging to clinical detection technical field.

Background technique

Prostaglandins (PGs) are a class of unsaturated hydroxy fatty acids with similar chemical composition and wide range of biological activities. The PGs family mainly includes: prostaglandin D ₂ , prostaglandin E ₂ , prostaglandin F _2α , prostaglandin I ₂ and TXA ₂ . The prostaglandin content in tissues is very small, but the activity is extremely strong. The endometrium is one of the important sites for the synthesis of prostaglandins. E ₂ and F _2α of prostaglandins are the main arachidonic acid products synthesized by the endometrium.

At present, prostaglandins are mostly detected by immunoaffinity chromatography-mass spectrometry (Immuno-GC-MS), liquid chromatography and mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS). The above detection methods can be quantitatively analyzed and have lower detection limits, but usually require expensive instruments and complicated operations, and long pretreatment and detection times, which seriously restrict the promotion of these detection methods, and cannot achieve large-scale rapid detection on site. Require. The immunoassay method has the characteristics of low cost, high throughput, high sensitivity, and relatively low requirements for technicians, so it is suitable for rapid screening of a large number of samples. The purpose of the present invention is to provide a method for preparing a monoclonal antibody hybridoma cell line with high affinity and detection sensitivity to prostaglandin F _2α , which lays the foundation for the development and promotion of indirect competitive ELISA kits and colloidal gold test strips.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an anti-prostaglandin F _2α -specific monoclonal antibody hybridoma cell line WXX-2, the antibody prepared from the cell line has good specificity and detection sensitivity to prostaglandin F _2α , and can be used for To establish an immunological detection method for prostaglandin F _2α .

The technical scheme of the present invention: an anti-prostaglandin F _2α -specific monoclonal antibody hybridoma cell line WXX-2, which has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration Commission, referred to as CGMCC, and the deposit number is CGMCC No. 13087.

The anti-prostaglandin F _2α -specific monoclonal antibody is secreted and produced by the anti-prostaglandin F _2α -specific monoclonal antibody hybridoma cell line WXX-2 with the deposit number of CGMCC No. 13087.

The application of the anti-prostaglandin F _2α specific monoclonal antibody in the clinical detection of prostaglandin F _2α .

The preparation steps of the anti-prostaglandin F _2α -specific monoclonal antibody hybridoma cell line WXX-2 are:

(1) Preparation and identification of immunogen: Prostaglandin F _2α was linked to the amino group of the protein carrier by the carbodiimide method (EDC method). After the reaction, the complete antigen and the unconjugated small molecule hapten were separated by dialysis. Complete antigens were identified by UV absorption scanning method;

(2) Immunization of mice: After the immunogen was completely emulsified with Freund's adjuvant, BALB/c mice were immunized by subcutaneous injection at multiple points. Freund's complete adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for booster immunization. The immunization dose during sprint immunization was half of the previous immunization dose. It was mixed with normal saline and injected directly into the abdominal cavity; the interval between each immunization was three weeks. . After the third immunization, blood was collected at intervals of one week to detect serum titer and inhibition;

(3) Cell fusion and establishment of cell lines: mouse splenocytes and mouse myeloma cells were fused by polyethylene glycol (PEG 2000) method, cultured in HAT medium, and positive cell wells were detected by indirect ELISA, and further The inhibitory effect of the positive cell wells was determined by indirect competitive ELISA, and the positive cell wells with the best inhibition were subcloned three times by limiting dilution method, and finally the anti-prostaglandin F _2α -specific monoclonal antibody hybridoma cell line WXX- 2;

(4) Identification of the properties of hybridoma cell lines: The mouse monoclonal antibody Ig class/subclass identification was determined with an enzyme-labeled secondary antibody kit; IC ₅₀ value and affinity were determined by ELISA.

The beneficial effects of the present invention: the anti-prostaglandin F _2α monoclonal antibody hybridoma cell line obtained by the present invention has better detection sensitivity and affinity for prostaglandin F _2α ; the present invention also provides a new synthetic prostaglandin F The method of _2α immunogen, the synthesis steps of hapten is more simplified and effective, which provides the idea and method of synthesizing immunogen for people's research in the future.

Preservation of biological material samples: monoclonal cell line WXX-2, which has been deposited in the General Microbiology Center of China Microbial Culture Collection Management Committee, referred to as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences , the preservation number is CGMCC No.13087, and the preservation date is October 31, 2016.

Description of drawings

Fig. 1 UV absorption spectroscopic characterization of immunogens.

Figure 2 Standard inhibition curve of anti-prostaglandin F _2α -specific monoclonal antibodies against prostaglandin F _2α .

Detailed ways

The following embodiments of the present invention are only used as a further description of the content of the present invention, and cannot be used as a limitation or scope of the present invention. The present invention will be further described below through examples.

In the present invention, mice are immunized with prostaglandin F _2α complete antigen, cultured in HAT selective medium through cell fusion, and the cell supernatant is screened through indirect ELISA and indirect competitive ELISA, and finally the prostaglandin F _2α with good affinity and high affinity is obtained. Sensitivity of monoclonal antibody hybridoma cell lines.

Example 1 Preparation of anti-prostaglandin F _2α -specific monoclonal antibody hybridoma cell line WXX-2

(1) Synthesis of complete antigen:

Synthesis of complete antigen: take 4 mg of hapten F _2α and add 5.0 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 3.7 mg of NHS (N-hydroxyl succinimide), dissolved in DMF (N,N-dimethylformamide), stirred at room temperature, and activated for 6h; another 15mg BSA (bovine serum albumin) was dissolved in 3mL, 0.05M, pH9.6 CBS ( Carbonate buffer solution) solution; add the above activation solution dropwise to the BSA solution, after stirring at room temperature overnight, remove the immunogen for PBS dialysis for 3 days, and store at -20°C.

(2) Animal immunization: healthy BALB/c mice aged 6-8 weeks were selected for immunization. The complete prostaglandin F _2α antigen (1 mg/mL) was emulsified with the same amount of Freund's adjuvant, and then BALB/c mice were immunized by subcutaneous injection at multiple points, each 100 μL. Freund's complete adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for booster immunization. The immunization dose during sprint immunization was half of the previous immunization dose. It was mixed with normal saline and injected directly into the abdominal cavity; the interval between each immunization was three weeks. . After the third immunization, blood was collected at an interval of one week to detect serum titer and inhibition; the mice with the best inhibition were selected and sprinted 18 days after the fifth immunization to prepare for fusion.

(3) Cell fusion: After three days of sprint immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight of 2000) method. The specific steps are as follows:

The mouse spleen was aseptically taken, ground and passed through a 200-mesh cell screen to obtain a spleen cell suspension, and the cells were counted;

Collect SP2/0 cells, suspend in RPMI-1640 basal medium, and count cells;

The splenocytes and SP2/0 cells were mixed according to the counting ratio of 2-10:1, and fused with PEG after centrifugation for 1 min. Then, RPMI-1640 basal medium was added from slow to fast. After centrifugation, suspended in 20% Fetal bovine serum and 2% 50×HAT RPMI-1640 screening medium were added to a 96-well cell culture plate and cultured in an incubator at 37°C and 5% CO ₂ .

(4) Cell selection and cell line establishment: On the third day of cell fusion, the fusion cells were screened with RPMI-1640 and the culture medium was half-changed, and on the fifth day, the cells were treated with 20% fetal bovine serum and 1% 100×HT. The RPMI-1640 transition medium was completely replaced, and the cell supernatant was taken for screening on the 7th day. The screening is divided into two steps: the first step is to screen the positive cells by indirect ELISA, and the second step is to select prostaglandin F _2α as the standard substance, and use indirect competitive ELISA to measure the inhibitory effect of positive cells. Cell wells with better inhibition of prostaglandin F _2α were selected, subcloned by limiting dilution method, and detected by the same method. Repeat three times to obtain anti-prostaglandin F _2α -specific monoclonal antibody hybridoma cell line WXX-2.

(5) Preparation and identification of anti-prostaglandin F _2α -specific monoclonal antibody: Take 8-10-week-old BALB/c mice, and each mouse was injected with 1 mL of paraffin oil; 7 days later, each mouse was injected with 1× Ascites was collected from 10 ⁶ hybridoma cells WXX-2 from the seventh day, the ascites was purified by the octanoic acid-saturated ammonium sulfate method, and the obtained monoclonal antibody was stored at -20°C.

The monoclonal antibody obtained by purification of ascites was subjected to immunoglobulin subtype identification using a mouse monoclonal antibody subtype identification kit, and its subtype was IgG1 type, as shown in Table 1.

Table 1 Identification of subtypes of prostaglandin F2α monoclonal antibodies

Using the indirect competitive ELISA method, the IC ₅₀ of the monoclonal antibody against prostaglandin F _2α was determined to be 1.8 μg/L, which can be used for the specific and rapid detection of prostaglandin F _2α , as shown in Figure 2.

(6) Antibody application: The monoclonal antibody prepared by the anti-prostaglandin F2α-specific monoclonal antibody hybridoma cell line WXX-2 through in vivo ascites was applied to the prostaglandin _F2α sample determination test. The specific steps are as follows:

6.1 Coat 0.1 μg/mL of prostaglandin F2α diluted with carbonate buffer (CBS) as the original coating. Coat 96-well microtiter plate, 100 μL per well, after coating for 2 hours at 37 °C, wash with PBST Wash the plate three times, 200 μL per well each time, 3 min each time, and pat dry;

6.2 Block with CBS containing 0.2% gelatin, 200 μL per well, block at 37°C for 2 hours, wash the plate three times with PBST washing solution, 200 μL per well for 3 min each time, and pat dry;

6.3 Prostaglandin F2α standard solution of 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 and 1 μg/L was prepared with phosphate buffered saline (PBS). Add the standard solution and the extract of the sample to be tested to the sealed microtiter plate, 50 μL per well, repeat 3 wells for each sample, and then add 50 μL 1:16000 diluted anti-prostaglandin F2α monoclonal to each well. Antibody, after 0.5h reaction at 37°C, wash the plate and pat dry;

6.4 Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted with PBS containing 0.1% gelatin at 1:3000 to each well, react at 37°C for 0.5 h, wash the plate and pat dry;

6.5 Add 100 μL of TMB chromogenic solution to each well, after 15 minutes of color development at 37°C, add 50 μL of 2M H ₂ SO ₄ stop solution to each well, and measure the absorbance at 450 nm.

Configuration of the solution:

Carbonate buffer solution (CBS): Weigh 1.59 g of Na ₂ CO ₃ and 2.93 g of NaHCO ₃ , dissolve them in a small amount of double-distilled water and mix them, add double-distilled water to about 800 mL and mix well, adjust the pH to 9.6, add Make up to 1000mL with double-distilled water and store at 4°C for later use;

Phosphate buffered saline (PBS): 8.00g NaCl, 0.2g KCl, 0.24g KH ₂ PO ₄ , 3.62g Na ₂ HPO ₄ ·12H ₂ O, dissolved in 800 mL of pure water, and adjusted to pH 7.2～7 with NaOH or HCl 7.4, dilute to 1000mL;

PBST: PBS containing 0.05% Tween20;

TMB color developing solution: Solution A: Na ₂ HPO ₄ ·12H ₂ O 18.43 g, citric acid 9.33 g, and purified water to 1000 mL; Solution B: 60 mg of TMB was dissolved in 100 mL of ethylene glycol. A and B liquids are mixed according to the volume ratio of 1:5, which is the TMB color developing liquid.

To sum up, the above are only preferred embodiments of the present invention, and are not intended to limit the scope of implementation of the present invention. That is, all equivalent changes and modifications made according to the content of the patented scope of the present invention shall fall within the technical scope of the present invention.

Claims (2)

1. Anti-prostaglandin F_2αThe specific monoclonal antibody hybridoma cell strain WXX-2 is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 13087.

2. Anti-prostaglandin F_2αA specific monoclonal antibody characterized by: the prostaglandin F resisting with the preservation number of CGMCC No.13087 as claimed in claim 1_2αAnd (3) secreting and producing a specific monoclonal antibody hybridoma cell strain WXX-2.

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